Strain-dependent effects on lung structure, matrix remodeling, and Stat3/Smad2 signaling in C57BL/6N and C57BL/6J mice after neonatal hyperoxia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm infants, characterized by lung growth arrest and matrix remodeling. Various animal models provide mechanistic insights in the pathogenesis of BPD. Since there is increasing evidence that genetic susceptibility modifies the response to lung injury, we investigated strain-dependent effects in hyperoxia (HYX)-induced lung injury of newborn mice. To this end, we exposed newborn C57BL/6N and C57BL/6J mice to 85% O-2 (HYX) or normoxia (NOX; 21% O-2) for 28 days, followed by lung excision for histological and molecular measurements. BL/6J-NOX mice exhibited a lower body and lung weight than BL/6N-NOX mice; hyperoxia reduced body weight in both strains and increased lung weight only in BL/6J-HYX mice. Quantitative histomorphometric analyses revealed reduced alveolar formation in lungs of both strains after HYX, but the effect was greater in BL/6J-HYX mice than BL/6N-HYX mice. Septal thickness was lower in BL/6J-NOX mice than BL/6N-NOX mice but increased in both strains after HYX. Elastic fiber density was significantly greater in BL/6J-HYX mice than BL/6N-HYX mice. Lungs of BL/6J-HYX mice were protected from changes in gene expression of fibrillin-1, fibrillin-2, fibulin-4, fibulin-5, and surfactant proteins seen in BL/6N-HYX mice. Finally, Stat3 was activated by HYX in both strains; in contrast, activation of Smad2 was markedly greater in lungs of BL/6N mice than BL/6J mice after HYX. In summary, we demonstrate strain-dependent differences in lung structure and matrix, alveolar epithelial cell markers, and Smad2 (transforming growth factor beta) signaling in neonatal HYX-induced lung injury. Strain-dependent effects and genetic susceptibility need be taken into consideration for reproducibility and reliability of results in animal models

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential

Dynamic Regulation of GH–IGF1 Signaling in Injury and Recovery in Hyperoxia-Induced Neonatal Lung Injury

Prematurely born infants often require supplemental oxygen that impairs lung growth and results in arrest of alveolarization and bronchopulmonary dysplasia (BPD). The growth hormone (GH)- and insulin-like growth factor (IGF)1 systems regulate cell homeostasis and organ development. Since IGF1 is decreased in preterm infants, we investigated the GH- and IGF1 signaling (1) in newborn mice with acute and prolonged exposure to hyperoxia as well as after recovery in room air; and (2) in cultured murine lung epithelial cells (MLE-12) and primary neonatal lung fibroblasts (pLFs) after treatment with GH, IGF1, and IGF1-receptor (IGF1-R) inhibitor or silencing of GH-receptor (Ghr) and Igf1r using the siRNA technique. We found that (1) early postnatal hyperoxia caused an arrest of alveolarization that persisted until adulthood. Both short-term and prolonged hyperoxia reduced GH-receptor expression and STAT5 signaling, whereas Igf1 mRNA and pAKT signaling were increased. These findings were related to a loss of epithelial cell markers (SFTPC, AQP5) and proliferation of myofibroblasts (αSMA+ cells). After recovery, GH-R-expression and STAT5 signaling were activated, Igf1r mRNA reduced, and SFTPC protein significantly increased. Cell culture studies showed that IGF1 induced expression of mesenchymal (e.g., Col1a1, Col4a4) and alveolar epithelial cell type I (Hopx, Igfbp2) markers, whereas inhibition of IGF1 increased SFTPC and reduced AQP5 in MLE-12. GH increased Il6 mRNA and reduced proliferation of pLFs, whereas IGF1 exhibited the opposite effect. In summary, our data demonstrate an opposite regulation of GH- and IGF1- signaling during short-term/prolonged hyperoxia-induced lung injury and recovery, affecting alveolar epithelial cell differentiation, inflammatory activation of fibroblasts, and a possible uncoupling of the GH-IGF1 axis in lungs after hyperoxia

Intrauterine growth restriction induces skin inflammation, increases TSLP and impairs epidermal barrier function

Intrauterine growth restriction (IUGR) and low birth weight are risk factors for childhood asthma. Atopic march describes the progression from early dermatitis to asthma during life. Since inflammatory signaling is linked to increased airway resistance and lung remodeling in rats after IUGR, we queried if these findings are related to skin inflammatory response. Firstly, we induced IUGR in Wistar rats by isocaloric protein restriction during gestation. IUGR rats showed lower body weight at postnatal day 1 (P1), catch-up growth at P21, and similar body weight like controls at P90. At P1 and P90, mRNA of inflammatory as well as fibrotic markers and number of skin immune cells (macrophages) were increased after IUGR. Skin thymic stromal lymphopoietin (TSLP) mRNA at P1 and serum TSLP at P1 and P21 were elevated in IUGR. Moreover, IUGR impaired transepidermal water loss at P21 and P90. IUGR induced higher. Secondly, the increase of TEWL after Oxazolone treatment as a model of atopic dermatitis (AD) was greater in IUGR than in Co. Our data demonstrate an early inflammatory skin response, which is linked to persistent macrophage infiltration in the skin and impaired epidermal barrier function after IUGR. These findings coupled with elevated TSLP could underlie atopic diseases in rats after IUGR. Key messages center dot The present study shows that IUGR increases macrophage infiltration and induces an inflammatory and fibrotic gene expression pattern in the skin of newborn rats. center dot Early postnatal inflammatory response in the skin after IUGR is followed by impaired epidermal barrier function later in life. center dot IUGR aggravates transepidermal water loss in an experimental atopic dermatitis model, possibly through elevated TSLP in skin and serum. center dot Early anti-inflammatory treatment and targeting TSLP signaling could offer novel avenues for early prevention of atopic disorders and late asthma in high-risk infants

Novel functional role of GH/IGF-I in neonatal lung myofibroblasts and in rat lung growth after intrauterine growth restriction

Intrauterine growth restriction (IUGR) is a risk factor for neonatal chronic lung disease (CLD) characterized by reduced alveoli and perturbed matrix remodeling. Previously, our group showed an activation of myofibroblasts and matrix remodeling in rat lungs after IUGR. Because growth hormone (GH) and insulin-like growth factor I (IGF-I) regulate development and growth, we queried 1) whether GH/IGF-I signaling is dysregulated in lungs after IUGR and 2) whether GH/IGF-I signaling is linked to neonatal lung myofibroblast function. IUGR was induced in Wistar rats by isocaloric low-protein diet during gestation. Lungs were obtained at embryonic day (E) 21, postnatal day (P) 3, P12, and P23. Murine embryonic fibroblasts (MEF) or primary neonatal myofibroblasts from rat lungs of control (pnF(Co)) and IUGR (pn(FIUGR)) were used for cell culture studies. In the intrauterine phase (E21), we found a reduction in GH receptor (GH-R), Stat5 signaling and IGF-I expression in lungs after IUGR. In the postnatal phase (P3-P23), catchup growth after IUGR was linked to increased GH mRNA, GH-R protein, activation of proliferative Stat5/Akt signaling, cyclin D1 and PCNA in rat lungs. On P23, a thickening of the alveolar septae was related to increased vimentin and matrix deposition, indicating fibrosis. In cell culture studies, nutrient deprivation blocked GH-R/IGF-IR signaling and proliferation in MEFs; this was reversed by IGF-I. Proliferation and Stat5 activation were increased in pn(FIUGR). IGF-I and GH induced proliferation and migration of pnF(Co); only IGF-I had these effects on pnFIUGR. Thus, we show a novel mechanism by which the GH/IGF-I axis in lung myofibroblasts could account for structural lung changes after IUGR